Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
13 Apr 2004
Deviations:
not specified
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 28 for the conduct of skin absorption studies, March 2004
Version / remarks:
March 2004
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Xyligen 30F is a 30% aqueous solution of K-HDO.
Specific details on test material used for the study:
NON-LABELED TEST SUBSTANCE:
Name of test substance: Xyligen 30 F
Test substance No.: 01/0069-7
Batch Identification: 44827924
CAS No.: 66603-10-9
Purity: Aqueous formulation; content of active ingredient K-HDO: 31.2%
Homogeneity: The test substance appeared to be homogeneous by visual inspection.
Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.

RADIOLABELED TEST SUBSTANCE:
Name of test substance: 14C-Reg. No. 5214771 (N-Cyclohexyldiazeniumdioxy-potassium)
Test substance No.: 05/0892-1
Batch Identification: 266-2001
Radiochemical purity: 98.9% (see Certificate of Analysis in Appendix 9.1).
Homogeneity: The homogeneity of the test substance was confirmed by analysis.

ADDITIONAL TEST SUBSTANCE INFORMATION:
IUPAC name: N-Cyclohexyldiazeniumdioxy-potassium
Label: K-HDO-U-C14
Specific activity: 3.66 MBq/mg a.i.

Radiolabelling:
yes

Test animals

Species:
other: not applicable, an in vitro test method was used
Strain:
not specified
Remarks:
not applicable, an in vitro test method was used
Details on test animals and environmental conditions:
Not applicable, an in vitro test method was used.

Administration / exposure

Type of coverage:
other: not applicable, an in vitro test method was used
Vehicle:
other:
Remarks:
not applicable, an in vitro test method was used
Doses:
see tables below
No. of animals per group:
not applicable
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: human donor
- Type of skin: Skin from abdomen
- Preparative technique: Split thickness skin preparations
- Supplier: Dermatomed human skin preparations with a thickness of 409 – 579 μm were supplied frozen by BIOPREDIC International, Rennes, France. On receipt they were vacuum-wrapped, labeled appropriately and stored frozen at –20°C until use.
- Thickness of skin (in mm): 409 – 579 μm
- Membrane integrity check: Before connecting the diffusion cell, equipped with skin preparation, to the experimental setup, the integrity of the skin preparation was determined by measuring its electrical resistance with a LCR bridge (LCR 400, Thurlby Thandar Instruments, England). For this purpose, about 0.8 mL physiological saline solution was pipetted onto the skin preparation and a stainless steel electrode (donor electrode diameter 2.05 mm) was suspended into the fluid column to a depth of about 0.5 cm. A further electrode (receptor electrode diameter about 0.95 mm) was introduced into the receptor compartment via the inlet of the Franz cell as far as possible. After the two electrodes were connected to the LCR 400 measuring bridge, the measured resistance was directly read on the display of the device. Measured values above 1 kOhm were expected for intact skin preparations (results are available in the raw data). In addition, skin preparation integrity was checked immediately before the application of the test substance preparation and immediately before the sampling procedures for balancing by visual inspection.
In the case of measured low resistance values, skin preparations were used unless the visual check revealed any damage. Further evaluation was based on the plausibility of results obtained for an individual skin preparation when compared to those obtained for the entire dose group. The following findings were reasons for not using a skin preparation in the study or rejecting results from calculation of the mean penetration parameters:
* A skin preparation showed low resistance and physical damage or leakage of receptor medium to the surface.
* A skin preparation displayed clearly aberrant test substance recovery rates (according to the guidelines of OECD a total recovery per membrane of 100 ± 10% is considered to be acceptable).
* A membrane displayed clearly aberrant penetration kinetics.

- Storage conditions: Skin preparations were stored for a maximum of 12 months at -20°C. Skin preparations were used no longer than the day after thawing in a refrigerator. Multiple freezing was omitted.
- Justification of species, anatomical site and preparative technique: Split thickness skin preparations were used to obtain as standardized test conditions as possible. This test system was used to determine the skin absorption of test substances in numerous investigations and represent a substantially validated model (see OECD Guidance Document No. 28). The preparations were supplied already dermatomized by the source stated above.

PRINCIPLES OF ASSAY
- Diffusion cell: Modified Franz cell consisting of an upper donor compartment and a wide opening at the top and a lower receptor compartment
- Receptor fluid: tap water
- Solubility od test substance in receptor fluid: The maximum solubility of the test substance in water is 542 g/L. Taking into account the maximum dose of active ingredient and the total volume of receptor fluid used including sampling, no rate limiting effects on the diffusion process by saturation of the receptor fluid is expected.
- Static system: The diffusion cells were operated in the static mode.
- Test temperature: 32 ± 1°C
- Occlusion: The donor compartment was covered with a sheet of Fixomull® Stretch (semiocclusive adhesive fleece).
- Other: The stability of the test substance in the test substance preparation over the test period was verified by analysis. The concentration and the homogeneous distribution of the test substance in the test substance preparation were confirmed by analysis.

Results and discussion

Absorption in different matrices:
See table below.
Total recovery:
See table below.

Any other information on results incl. tables

Mean recovery rates from the different compartments of the diffusion cell

Nominal Dose of Test Substance [μg]

3120

 

Mean applied Dose of Test Substance [μg]

3194

 

 

 Dose of Test Substance  

Found [μg]

% of Applied Dose

Non-absorbed Dose

 

 

Tape strips

69.8

2.18

Membrane Washing

2566.5

80.37

Sum

2636.3

82.55

Skin preparation

92.9

2.90

Absorbed Dose

 

 

Sum Receptor Samples 0 - 24 h Including Wash Out

93.4

2.93

Receptor Fluid

358.9

11.22

Receptor Chamber Washing

7.0

0.22

Sum

459.3

14.37

Total

3188.5

 

Total Recovery

 

99.83

 

The mean total recovery measured in the diffusion cells fulfilled the OECD quality criteria.

The individual values ranged between 98 and 101%. The major amount of supplied test substance was recovered from the skin membrane washings and tape strips (83%). The individual values ranged between 72.5 and 86.9%. About 3% of the test substance was associated with the skin membrane. The individual values ranged between 1.3 and 4.8%. About 14% were recovered from the receptor fluid and receptor compartment, which is considered to represent the absorbed dose. The individual values ranged between 9.2 and 21.6%.

Nominal Dose of Test Preparation [mg/cm²]

10

Nominal Dose of Test Substance [µg/cm²]

3120

Applied Dose of Test Substance [µg/cm²]

3194

Sample Time [h]

Mean Cumulative Absorption of the Test Substance [µg/cm²]

Percentage Absorption

0.5

1.55

0.05

1

4.87

0.15

2

13.24

0.42

4

38.02

1.19

6

75.45

2.37

10

159.34

5.00

24

435.49

13.62

 

 

 

Kp [*10-5cm/h]

8.81

 

Absorption Rate [µg / (cm²*h)]

27.98

 

Lag Time [ h ]

4.13

 

Applicant's summary and conclusion

Conclusions:
In conclusion, the total recovery rates of the test substance show the validity of the experimental results. According to the categorization schemes suggested by Marzulli et al. 1969 and Barber et al. 1995 the permeability constant (Kp) shows a moderate penetration of 14C-Reg. No. 5214771 (N-Cyclohexyldiazeniumdioxy-potassium) through human skin preparations under the test conditions used.
Executive summary:

The dermal penetration of the active ingredient through human skin in vitro was examined after application of a formulation concentrate to dermatomized skin preparations mounted on Franz-type diffusion cells.

The target dose of about 10 mg or μL/cm² corresponding to about 3120 μg/cm² of the test substance formulation was achieved well.

The total recovery rates range between 98.2 and 101.3% of the applied dose, which is fully in compliance with the quality criteria of the OECD Guideline.

The major amount of the test substance was recovered from the skin membrane washings and tape strips. This non-absorbed dose was about 82.6%.

The mean relative amount of test substance associated with the skin preparation was 2.9%. The relative mean absorbed dose, i.e. the amount of test substance found in the samples of the receptor fluid and derived from the receptor compartment washes in relation to the applied dose, was 14.4%. This indicates some diffusion occurs across the skin membranes.

The permeability constant was calculated from the absorption rate (about 28 μg/(cm²*h) and the concentration of the applied test substance preparation to be 8.8*10-5 cm/h. The region of the steepest slope of individual absorption-time curves was used for linear regression analysis. Thus the value represents the maximum penetration constant. According to the categorization schemes of Marzulli et al. 1969 and Barber et al. 1995 the permeability constant (Kp) shows a moderate penetration of the test substance through human skin under the test conditions used.

The mean absorption lag time was calculated to be about 4 hours. The presence of a lag time of absorption underlines the functional diffusion barrier of the skin against the tested substance preparation.